Diagnostic reagent for syphilis

ABSTRACT

A REAGENT AND METHOD FOR THE SEROLOGICAL DIAGNOSIS OF SYPHILIS BY DETERMINING THE AGGLUTINATION OF ANTIGEN-COATED LATEX PARTICLES WITH A PATIENT&#39;&#39;S SERUM IN WHICH THE REAGENT USED IN THE DIAGNOSIS CONSISTS OF POLYSTYRENE LATEX HAVING A PARTICLE SIZE OF FROM ABOUT 0.1 TO ABOUT 0.35 MICRON COATED WITH A COMBINATION OF FROM ABOUT 0.007 MG. TO ABOUT 0.187 MG. OF CARDIOLIPIN AND FROM ABOUT 0.0005 MG. TO ABOUT 0.03 MG. REITER PROTEIN ANTIGEN PER MG. OF LATEX SOLIDS.

United States Patent 3,564,089 DIAGNOSTIC REAGENT FOR SYPHILIS Sandra Jean Kiddy, 1245 Doremus Road, Pasadena, Calif. 91105 No Drawing. Filed Sept. 29, 1966, Ser. No. 583,065 Int. Cl. G01n 31/00, 31/02, 33/16 US. Cl. 424-13 1 Claim ABSTRACT OF THE DISCLOSURE This invention relates to a diagnostic reagent and test for syphilis. More particularly, this invention is concerned with the use of latex particles in a serologic test for syphilis in which the latex particles are coated with two ditferent antigens.

At the present time there are numerous serologic tests which have been developed for the diagnosis of syphilitic infection. Serologic Tests for Syphilis, 1964 Manual, Public Service Publication No. 411 (rev. ed. 1964). Many of these diagnostic tests are complicated and time consuming.

A complete serologic diagnosis for syphilis includes both a nontreponemal test and a test for treponemal antibody. The nontreponemal tests include, e.g., the Wassermann and Kolmer complement fixation tests, as well as the microflocculation tests of Kahn, Kline, Eagle, Hinton, Mazzini and VDRL. Tests for treponemal antibody include the Reiter Protein Complement Fixation test and other procedures such as those described, e.g., by Deacon and Hunter, Proc. Soc. Exper, Biol, and Med, vol. 110, pp. 352-56 (1962).

Recently, a serologic test for syphilis was developed which employs latex particles as a vehicle for cardiolipin antigen. The agglutination of the cardiolipin-coated latex particles with both VDRL- and Kolmer-positive sera demonstrated sensitive activity, Uyeda, Amer. J. Clin. Path., vol. 40, pp. 329-33 (1963). The test employed polystyrene latex particles of 0.81 micron in diameter.

In another recent development of a serologic test for syphilis, polystyrene latex particles of 0.79 micron in diameter were employed as a vehicle for Reiter protein antigen. This agglutination test for treponemal antibody compared favorably with the Reiter Protein Complement Fixation test, Stevens, Amer. J. Clin. Path., vol. 43, pp. 490-93 (1965).

In accordance with the present invention, a diagnostic reagent and test for syphilis is provided in which a complete diagnosis for syphilis, i.e., for both treponemal and nontreponemal antibodies, is obtained with a single test reagent unit which is rapid and simple in operation. In brief, the diagnostic reagent of this invention comprises polystyrene latex having a particle size-in the range of about 0.10 to about 0.35 micron coated with a combination of cardiolipin and Reiter protein antigens.

The polystyrene latex vehicle used in the diagnostic reagent of this invention is a high molecular weight polymer which is produced by polymerizing styrene monomer in the presence of water to form lattices. The polymer particles are in the dispersed phase in an aqueous suspension and are negatively charged. Polystyrene latex is stable 3,564,089 Patented Feb. 16, 1971 to repeated cycles of freezing and thawing and is infinitely dilutable in water. The latex appears to be self-sterilizing.

Suitable examples of polystyrene latex suspensions are materials available from Monsanto Chemical Company under the trademark Lytron 615 and from Koppers Company, Inc. under the trademark Dylex K-31.

The polystyrene latex suspension used in the diagnostic reagent of this invention contains on the order of about 3% to 5% solids, and preferably about 4% solids. If the commercially available product is more concentrated, it can be diluted for purposes of this invention by conventional means to the proper concentration with sterile distilled water.

The two antigens which are used according to the present invention to coat the polystyrene latex particles have been described elsewhere. Cardiolipin is a phospholipid obtained from beef heart. Its preparation and composition are described by Pangborn, Proc. Soc. Exp. Biol; and Med., vol. 48, pp. 484-86 (1941) and J. Biol. Chem., vol. 143, pp. 247-56 (1942), vol. 153, pp. 343-48 (1944), vol. 161, pp. 71-82 (1945) and vol. 168, pp.

Cardiolipin antigen generally consists of a mixture of cardiolipin, cholesterol and lecithin. One such composition comprises:

Percent Cardiolipin 0.03 Cholesterol 0.9 Lecithin 0.21 10.01

This composition is known as the VDRL antigen and is described in Serologic Tests for Syphilis, 1964 Manual, supra. Other cardiolipin antigen compositions are those described, e.g., by Mazzini (Serologic Tests for Syphilis, 1964 Manual, supra.)

It will be understood that the above and similar cardiolipin antigen compositions for testing nontreponemal antibodies are included within the scope of this invention.

Reiter protein is prepared from a culture of Reiter treponemes by cryolysis and ammonium sulfate precipitation. Its preparation is described by DAlessandro and Dardanoni, Amer. J. Syph., vol. 37, pp. 137-50 (1953). Reiter protein is used in the Reiter Protein Complement Fixation test. The actual reactive substance has not been defined. The optimal dilution of antigen for use in the test is determined by the reactivity of various dilutions with known human syphilite sera. Under these circumstances the milligrams of protein present per ml. of antigen solution may vary over a Wide range. Reactivity of the antigen is not directly proportional to the total amount of protein present. In practice, the optimal dilution of commercially-available antigen for use in the complement fixation test as stated on the label is 1:60. Standardized Reiter protein is obtainable commercially from Sylvana Chemical Company and Lederle Laboratories. The total protein concentration in the standardized antigens obtained from these companies was determined by spectrophotometry. The antigen from Lederle contained 0.16350 mg. protein per ml. of undiluted solution, while that from Sylvana contained 1.0536 mg. protein per ml. of undiluted solution.

In the practice of the present invention, the polystyrene latex suspension and the cardiolipin and Reiter protein antigens are admixed in an aqueous buffered solution. Normally, the pH of the buffered solution is above about 8.0, and preferably at about 8.2. The buffered solution also preferably contains glycine and saline. Heating of the reagent speeds up the process of coating. The adsorption of cardiolipin to the latex particle is accomplished more rapidly by heating to 5558 C. for several hours than if allowed to stand at room temperature. Reiter protein, however, is unstable at this temperature; therefore, coating of this antigen is carried out at 37 (35-39 C.).

The concentration of the antigens in the latex reagent varies from about 0.007 mg. to about 0.187 mg. of cardiolipin per mg. of latex solids and from about 0.0005 mg. to about 0.03 mg. Reiter protein per mg. of latex solids. A preferred embodiment of the invention contains about 0.1 mg. of cardiolipin per mg. of latex solids and about 0.004 mg. Reiter protein per mg. of latex solids. The standardized Reiter protein in its undiluted form is present in an amount by volume of from about 0.005 ml. to about 0.05 ml. per mg. of latex solids. The volume to weight relationship of Reiter protein to latex is the preferred method of stating the concentration of this antigen in the latex.

The diagnostic reagent and test described herein provides the following advantages in practice compared to conventional diagnostic tests for syphilitic infection:

(1) It is ready to use and does not require further preparation.

(2) It is a stable reagent and eliminates the need for preparing fresh antigen daily. It is stable for 6 months at a storage temperature of 210 C.

(3) There is no need to standardize the antigen against known positive and negative sera in clinical laboratories.

(4) The use of this reagent does not require special equipment such as needles, microscopes, special pipettes, etc., required in conventional syphilis diagnostic procedures.

(5) The test results are rapidly obtained, i.e., within about two minutes after the initial heat inactivation of the serum.

The following examples further illustrate the invention described and claimed herein although the invention is not limited to these specific examples.

EXAMPLE I This example illustrates the preparation and stability of the diagnostic reagent of the present invention. In this example, a polystyrene latex suspension known as Lytron 615, obtained from Monsanto Chemical Company, is employed in sterile form. The suspension is first centrifuged and the packed particles resuspended in an equal volume of sterile distilled water.

The cardiolipin antigen employed in this example is the VDRL antigen obtained from Lederle. It is reported to have a composition of 0.03% cardiolipin, 0.9% cholesterol, and 0.21:0.0l% lecithin.

The Reiter protein of this example was obtained from Lederle. The dilution for use in the complement fixation test was 1:60.

In the use of the above materials, two aqueous buffered solutions are prepared as follows:

Buffer A Adjust to pH 8.2 with NaOH:

0.05 M glycine 0.85% NaCl 0.00005 M EDTA (ethylenediaminetetraacetic acid) Buffer B:

0.71 M choline chloride added to Buffer A-no further adjustment in pH.

The above concentrate of choline chloride can be varied from about 0.55 M to about 0.85 M.

The diagnostic reagent of this example is prepared as follows:

(1) Precipitate 150 ml. cardiolipin antigen with 300 ml. Buffer A. The ratio of Buffer A to antigen can be varied from 0.5 to an infinite number of volumes of Buffer A to one volume of antigen. The importance of this step is to remove the antigen from the alcoholic solution in which it is commercially obtained.

(2) Centrifuge at 13,000 gravity for 20 minutes. Pour off supernatant.

(3) Add 10 to 20 ml. Buffer A or enough to thoroughly resuspend precipitate.

(4) Add 10 ml. latex which had been previously centrifuged and resuspended in an equal volume of water.

(5) Make up to 200 ml. with Buffer A.

(6) Heat in 56 C. water bath for 4 hours or hold at 5 C. for 12 to 24 hours.

(7) If heated, cool to room temperature after removing from water bath and refrigerate for 48 hours (2 10 C.).

(8) If held at 5 C., hold for an additional 48 hours.

(9) Bring to room temperature.

(10) Add 2 ml. Reiter protein and stir 16 to 24 hours at 37 C.

(11) Refrigerate for 48 hours (2-10 C.).

(12) Centrifuge at 13,000 gravity for 30 to 45 minutes and resuspend to equal volume (200 ml.) in Buffer B.

(13) Refrigerate 48 hours before use (210 C.).

(14) If the sensitivity of the reagent is low, additional Reiter protein may be added by following directions 10- 13. This may be repeated as often as necessary until the reagent has optimal sensitivity.

In the above example, the concentrations of the antigens are calculated as follows:

Cardiolipin.Based on the above-described composition of the cardiolipin and the use of the latex having 4% solids:

0.03% cardiolipin-0.03 gm./ ml. solution or 0.3

mg./ml.

0.9% cholesterol0.9 gm./100 ml. solution or 9.0 mg./

0.21% lecithin-0.21 gm./100 ml. solution or 2.1 mg./

In the above procedure, 15 ml. of cardiolipin antigen are used per ml. of latex particles. Therefore, the number of milligrams per ml. cardiolipin antigen used for each ml. of latex should be multiplied by 15.

cardiolipin-0.3 mg./ ml. 15 :45 mg./ml. of latex Cholesterol9.0 mg./ml. l5=135.0 mg./ml. of latex Lecithin2.1 mg./ml. 15=3.15 mg./ml. of latex Latex concentration4% solids or 4.0 gm./100 ml. or

40 mg./ml.

Therefore, the concentration of cardiolipin per mg. latex 1s:

cardiolipin-4.5 mg./ml.+40 mg./ml.=0.112 mg./mg.

latex Cholesteroll35.0 mg./ml.-:40 mg./ml.=3.375 mg./

mg. latex Lecithin3l.5 mg./ml.-:40 mg./ml.=0.7875 mg./mg.

latex In the above example, 15 ml. of Mazzini antigen (prepared per m1. stock latex) and 5 ml. of Kolmer antigen (prepared per ml. of stock latex) can be substituted for the VDRL antigen to provide a substantially equivalent diagnostic reagent for syphilis.

Reiter protein.In the preparation of the above diagnostic reagent, the optimal concentration of undiluted Reiter protein antigen bearing the titer on the label was found to be 1 ml. per 100 ml. of reagent or 1 ml. per 5 ml. of latex.

The diagnostic reagent of this example was found to be stable after being stored for six months at a storage temperature of 10 C.

EXAMPLE II This example illustrates the convenient use and reliability of the diagnostic reagent of the invention defined herein in the diagnosis of syphilis. The following procedure is used:

(1) Heat test serum at 56 C. for 30 minutes.

(2) Remove diagnostic reagent as prepared in Example I from refrigerator and warm to room temperature by standing in air.

(3) Using a capillary pipette, place 2 drops of heated serum in the center of a ringed area on a clean glass slide.

(4) Add 1 drop of diagnostic reagent and mix well with a wooden applicator stick.

(5) Tilt for 2-4 minutes or rotate for 2 minutes on a rotator similar to that type approved for the VDRL Microflocculation Slide Test.

(6) Read against a black background using a fluorescent light or other White light at an angle above the slide. Agglutination of the latex particles is compared with that found in known positive and negative human syphilitic sera.

Negativeno agglutination or agglutination not stronger than that seen in the negative control Positive-small or large clumps or aggregates of latex particles with a cloudy or clear background Tests were run by two independent cooperating laboratories on 565 unknown sera. The results of these tests comparing VDRL Microfiocculation Slide Test results with those of the diagnostic reagent of the present invention are shown in the accompanying Table I. In this table the term Latex STS (screening test for syphilis) referes to the tests employing the diagnostic reagent of the present invention. The terms R, WR and NR are official designations for VDRL test results (Serologic Tests for Syphilis, 1964 Manual, supra) which refer to reactive, weakly reactive and non-reactive results, respectively, in that test. The 514 sera testing negative and nonreactive in the first cooperating laboratory were not re TABLE I peated by the second cooperating laboratory. The remaining 51 were retested except one which contained insufiicient serum to repeat both tests. The terms Correlation and Non-Correlation refer to the comparison of VDRL test results with those of Latex STS. They do not refer to the correlation of results between the two laboratories. The correlation between the VDRL test results and the Latex STS results in this example is about 93% (529/565).

EXAMPLE III This example illustrates a preferred embodiment of the present invention, although it will be understood that the reagent can be packaged in other forms, e.g., with less than all of the stated materials, or in combination with other materials, or in other proportions.

The diagnostic reagent is packaged in a kit form. This kit includes 5 ml. of the diagnostic reagent of the present invention, 1 ml. of positive control serum, 1 ml. of negative control serum, 100 capillary pipettes for delivering test specimen and 1 divided glass slide containing 9 rings for performance of the test.

As will be readily apparent to those skilled in the art, other examples of the herein defined diagnostic reagent and test for syphilis can be devised by various modifica tions, variations and adaptations without departing from the spirit and scope of the invention after reading the foregoing specification and the claim appended hereto. All such modifications, variations and adaptations are included within the scope of the invention as defined in the appended claim.

What is claimed is:

1. A reagent for the diagnosis of syphilis comprising polystyrene latex having a particle size of from about 0.1 to about 0.35 micron coated with beef healt cardiolipin antigen comprising cardiolipin, cholesterol and lecithin and adjusted to from about 0.007 mg. to about 0.187 mg. of cardiolipin per mg. of latex solids and from about 0.0005 mg. to about 0.03 mg. Reiter treponemal protein antigen per mg. of latex solids.

100% correlation Non-correlation 1st cooperating laboratory 2nd cooperating laboratory 1st cooperating laboratory 2nd cooperating laboratory Num- Num- Nnm- Num- Latex STS VD RL ber Latex STS VDRL ber Latex STS VDRL ber Latex STS VDRL ber Positive R 5 Positive R 9 Negative R 0 Negative R 0 Do WR 10 .do WR 7 do WR 17 do WR 2 Negative NR 514 Negative NR 15 Positive NR 19 Positive* NR 17 No test was run-insutfieient sera.

References Cited UNITED STATES PATENTS 5/1963 Fisk 42412 1/1963 Brewer 424-13 OTHER REFERENCES Faure: Annales de lInstitut Pasteur, vol. 99, No. 5,

De Almeida: JAMA, 156:3, Sept. 18, 1954. p. 290. DAlessandro: Amer. J. of Syphilis, vol. 37, 1953, pp.

STANLEY J. FRIEDMAN, Primary Examiner A. P. FAGELSON, Assistant Examiner US Cl. X.R.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3, 089 D t d February 16, 1971 Inventor) Sandra Jean Kiddy It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

In the heading, at col. 1, line 4, after "91105" insert --assignor to Hyland Laboratories-- Signed and sealed this 8th day of June 1971.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. Attesting Officer WILLIAM E. SCHUYLER, JR. Commissioner of Patents 

